Cold-active serine alkaline protease from the psychrophilic bacterium Pseudomonas strain DY-A: enzyme purification and characterization
Tóm tắt
An extracellular protease was purified from a deep-sea psychrophilic bacterium strain DY-A which was identified as a Pseudomonas species. The optimal growth and protease-producing temperatures of the strain were all 10°C, and the protease was secreted only at temperatures under 20°C. The enzyme was most active at 40°C and at pH 10.0. It was inhibited by phenylmethyl sulfonylfluoride and diisopropyl fluorophosphate, indicating that it is a serine protease. Chelators such as EDTA, EGTA, 1,10-phenanthroline and 2,2′-bipyridyl produced a decrease of activity. The enzyme was sensitive to denaturing agents such as SDS, urea, and guanidine HCl and resistant to thiol-containing reducing agents such as dithiotreitol. The enzyme was active towards N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide and N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide. The native molecular mass of the enzyme determined by native PAGE and SDS-PAGE was 25 kDa.
Tài liệu tham khảo
Chessa JP, Petrescu I, Bentahir M, Van Beeumen J, Gerday C (2000) Purification, physico-chemical characterization and sequence of a heat labile alkaline metalloprotease isolated from a psychrophilic Pseudomonas species. Biochim Biophys Acta 1479:265–274
Feller G, Narinx E, Arpigny JL, Aittaleb M, Baise E, Genicot S, Gerday C (1996) Enzymes from psychrophilic organisms. FEMS Microbiol Rev 18:189–202
Gerday C, Aittaleb M, Arpigny JL, Baise E, Chessa JP, Garsoux G, Petrescu I, Feller G (1997) Psychrophilic enzymes: a thermodynamic challenge. Biochim Biophys Acta 1342:119–131
Hoshino T, Ishizaki K, Sakamoto T, Kumeta H, Yumoto I, Matsuyama H, Ohgiya S (1997) Isolation of a Pseudomonas species from fish intestine that produces a protease active at low temperature. Lett Appl Microbiol 25:70–72
Kulakova L, Galkin A, Kurihara T, Yoshimura T, Esaki N (1999) Cold-active serine alkaline protease from the psychrotrophic bacterium Shewanella strain Ac10: gene cloning and enzyme purification and characterization. Appl Environ Microbiol 65:611–617
Margesin R, Plama N, Knauseder F, Schinner F (1991) Proteases of psychrotrophic bacteria isolates from glaciers. J Basic Microbiol 31:377–383
Vazquez SC, Rios Merino LN, MacCormack WP, Fraile ER (1995) Protease-producing psychrotrophic bacteria isolated from Antarctica. Polar Biol 15:131–135